Transfection of autologous host cells in vivo using gene activated scaffolds incorporating star-polypeptides

It is increasingly being recognised within the field of tissue engineering that the regenerative capacity of biomaterial scaffolds can be augmented via the incorporation of gene therapeutics. However, the field still lacks a biocompatible gene delivery vector which is capable of functionalizing scaffolds for tailored nucleic acid delivery. Herein, we describe a versatile, collagen based, gene-activated scaffold platform which can transfect autologous host cells in vivo via incorporation of star-shaped poly(˪-lysine) polypeptides (star-PLLs) and a plasmid DNA (pDNA) cargo. Two star-PLL vectors with varying number and length of poly(˪-lysine) arms were assessed. In vitro, the functionalization of a range of collagen based scaffolds containing either glycosaminoglycans (chondroitin sulfate or hyaluronic acid) or ceramics (hydroxyapatite or nano-hydroxyapatite) with star-PLL-pDNA nanomedicines facilitated prolonged, non-toxic transgene expression by mesenchymal stem cells (MSCs). We demonstrate that the star-PLL structure confers enhanced spatiotemporal control of nanomedicine release from functionalized scaffolds over a 28-day period compared to naked pDNA. Furthermore, we identify a star-PLL composition with 64 poly(˪-lysine) arms and 5 (˪-lysine) subunits per arm as a particularly effective vector, capable of facilitating a 2-fold increase in reporter transgene expression compared to the widely used vector polyethylenimine (PEI), a 44-fold increase compared to a 32 poly(˪-lysine) armed star-PLL and a 130-fold increase compared to its linear analogue, linear poly(˪-lysine) (L-PLL) from a collagen-chondroitin sulfate gene activated scaffold. In an in vivo subcutaneous implant model, star-PLL-pDNA gene activated scaffolds which were implanted cell-free exhibited extensive infiltration of autologous host cells, nanomedicine retention within the implanted construct and successful host cell transfection at the very early time point of just seven days. Overall, this article illustrates for the first time the significant ability of the star-PLL polymeric structure to transfect autologous host cells in vivo from an implanted biomaterial scaffold thereby forming a versatile platform with potential in numerous tissue engineering applications.